Systems and methods for cryopreservation of cells

ABSTRACT

An auto-nucleating device includes a tube containing a crystalline cholesterol matrix. The ends of the tube are closed by a membrane impermeable to the cholesterol but permeable to liquids contained in a cryopreservation vessel. The auto-nucleating device provides a site for ice nucleation during freezing of the liquid within the vessel. The cryopreservation vessel can be a flexible vial having a closed port adapted to be pierced by a needle, and an opposite end that is initially open to receive the liquid. Another vessel includes an adaptor mounted to liquid container with a tubular branch closed by a needle septum and another tubular branch provided with a barbed fitting for engaging a flexible tube that terminates in a needle septum. Alternatively, the vessel includes an inlet and vent branch at the top of the container and an outlet septum at a bottom opening.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a continuation of U.S. patent applicationSer. No. 12/337,237, filed Dec. 17, 2008, now U.S. Pat. No. 8,222,027,issued Jul. 17, 2012, which is a continuation-in-part and claimspriority to co-pending U.S. patent application Ser. No. 11/765,000, nowU.S. Pat. No. 7,939,316, issued May 10, 2011, and to internationalpatent application No. PCT/US2007/071545, both of which were filed onJun. 19, 2007, and both of which claim the benefit of U.S. ProvisionalApplication No. 60/814,982, entitled “Systems and Methods forCryopreservation of Cells”, which was filed on Jun. 20, 2006, all ofwhich are incorporated herein by reference.

BACKGROUND OF THE INVENTION

The present invention concerns storage methods and associated devicesfor cryopreservation of cells, such as mammalian cells, and tissuesamples/specimen.

Cells and tissues are frequently cryopreserved to temporally extendtheir viability and usefulness in biomedical applications. The processof cryopreservation involves, in part, placing cells into aqueoussolutions containing electrolytes and chemical compounds that protectthe cells during the freezing process (cryoprotectants). Suchcryoprotectants are often small molecular weight molecules such asglycerol, propylene glycol, ethylene glycol or dimethyl sulfoxide(DMSO).

As these solutions are cooled to temperatures slightly below theirfreezing point, the solution remains in the liquid state. This conditionin which the solution remains liquid below its phase transitiontemperature is termed supercooling. As the aqueous solutions are cooledfurther below their freezing point, the extent of supercoolingincreases. In the absence of intervention, the water molecules in thesolution will, at a point usually no more than 15° C. below the freezingpoint, spontaneously crystallize, and pure water will precipitate asice.

During this transition from the liquid to the solid state, the solutionmoves from a higher to a lower free energy state, resulting in anexothermic reaction. The heat produced during this phase transitioncauses a transient warming of the sample during which the sampletemperature increases. Meanwhile the surrounding environment (e.g. thedevice in which the sample is being cryopreserved) either remains at aconstant temperature or continues to cool (depending upon the coolingapproach used). Subsequently, as the heat in the sample dissipates, thethermal disequilibrium between the sample and cooling device createdduring this event causes the sample to undergo a rapid cooling rate tore-establish thermal equilibrium. In many cases this rapid cooling ratecauses the formation of intracellular ice, which usually results in celldeath. This formation of intracellular ice is typically dependent uponthe mass of the sample, the heat transfer properties of the samplecontainer, the cooling protocol used and the fundamental cryobiologicalproperties of the cells.

The relationship between the frozen state and living systems has beenfascinating mankind for years. As early as 1683, Robert Boyle observedthat some fish and frogs could survive sub-freezing temperatures forshort periods of time if a fraction of their body water remainedunfrozen. Artificially induced cryopreservation was first observed in1948 by Polge, Smith, and Parkes by the serendipitous discovery of thecryo-protective properties of glycerol for fowl and bull semen and,subsequently, for red blood cells. In more recent times, scientistsinterested in the natural phenomena and biomedical applicationsassociated with freezing biological systems have begun to investigatethe fundamental processes governing the relationship. To begin with, itis well known that decreased temperature results in the suppression ofmetabolic activity and, thus, in a reduction of the rate at whichdeterioration of an unnourished biological system would occur. Thefreezing process, however, is not as benign as one might assume; itgenerally induces extreme variations in chemical, thermal, andelectrical properties that could be expected to alter intracellularorganelles, cellular membranes and the delicate cell-cell interactionsystems associated with tissues and organs. Indeed, given the extremecomplexity of even the simplest biological cells, it is thereforeremarkable that a reversible state of suspended animation by freezing ispossible at all.

Since that first discovery of the cryoprotective effects of glycerol andthe subsequent discovery of the widely applicable permeatingcryoprotectant dimethyl sulfoxide (DMSO)), many investigators haveattempted the preservation of cells or tissues, mostly through empiricalmethods. Most cell suspension cryopreservation protocols have beenestablished using molar concentrations of permeating cryoprotectiveadditives to enable freezing survival. By using these artificialcryoprotectants, much flexibility has been added to the cryopreservationprocess. For example, human red blood cells need to be cooled at a rateof around 1000° C./min. for optimal survival without the addition of acryoprotective agent (CPA). In the presence of 3.3M (30%) glycerol,however, survival of this cell type remains around 90% over a 2-3 logrange in cooling rates. As can be expected, the higher the CPAconcentration, the greater the likelihood of osmotic damage during theaddition/removal of the substance, and consequently the greater carethat is necessary in these processes.

During any cryopreservation process, the solutions involved willsupercool below their freezing point until they find a random nucleationsite for crystal formation. When cryopreserving by a freeze-thaw method,ice formation in the extracellular medium should be deliberatelyinitiated by seeding at low degrees of supercooling. If ice formation isnot induced by seeding, ice will form spontaneously when the solution iscooled sufficiently far below its equilibrium freezing point. Becausethis process is random in nature, ice formation will occur at random,unpredictable temperatures; consequently, sample survival rates will behighly variable between repeated trials with the same freezing protocol.Furthermore, the extremely rapid crystallization which results when iceforms in a highly supercooled solution causes damage to cells andtissues. Moreover, it has been shown that if extracellular ice formationis initiated at high degrees of supercooling, the probability ofdamaging intracellular ice formation is drastically increased. Thisphenomenon results from the delayed onset of freeze-induced celldehydration, which results in increased retention of intracellularwater, and thus higher likelihood of ice formation in the cell.

As noted above, during the transition from the liquid to the solidstate, the solution moves from a higher to a lower free energy statewhich results in thermal disequilibrium between the sample thatcontinues to warm and the cooling device that continues to cool. Thisdisequilibrium ultimately results in a severe deviation from the coolingrate prescribed for the particular cell type, and the potential for celldamage during the process.

To prevent these potentially damaging situations from occurring, stepsin the cryopreservation process often include interventions to introduceice crystals in the extracellular solution near the solution freezingpoint. This process called “seeding” is typically performed by coolingthe samples to near the solution freezing point, then touching theoutside of the sample container with a metal device (e.g. forceps or ametal rod) precooled in a cryogenic fluid (e.g. liquid nitrogen). Thisseeding step produces ice crystals in the extracellular solution andprovides a “template” upon which supercooled water molecules in thesolution organize and produce further ice. However, seeding samples inthis manner is time consuming and places the samples at risk in caseswhere they are temporarily removed from the cooling device for thisprocedure and because this method of seeding may inadvertently causeintracellular ice formation.

There is a need for a cryopreservation system that avoids the problemsassociated with the disequilibrium conditions described above. There isa further need for such a system that does not require the ancillaryseeding step currently conducted to induce controlled ice crystalproduction. There is an additional need for a cryopreservation devicethat facilitates the solution to the above-noted problems. The neededcryopreservation device should also provide means to simplify its use inacquiring and storing cells and tissue to be cryopreserved.

SUMMARY OF THE INVENTION

These and other needs in the field of cryopreservation are met byseveral aspects of the present invention. In one aspect of theinvention, an auto-nucleating device is provided for introduction into acryopreservation vessel prior to freezing of a liquid contained therein.The device comprises an elongated hollow tube sized for introductioninto the cryopreservation vessel and an ice-nucleating compositiondisposed within the hollow tube. Both ends of the tube are sealed, whileat least one end is sealed with a membrane that is impermeable to theice-nucleating composition but permeable to the liquid contained withinthe cryopreservation vessel. Preferably, both ends include the membraneto permit flow of the sample liquid into and through the device.

In the preferred embodiment, the ice-nucleating composition is a sterol,and most preferably cholesterol. The cholesterol may be a coating on theinterior of the hollow tube or may be provided as a solid matrix withinthe tube.

In another aspect of the invention, cryopreservation vessels areprovided that may be used with the auto-nucleating device. In oneembodiment, the cryopreservation vessel comprises a flexible tubularbody having one end initially open for the introduction of a liquidsample into the body and a closed port defined at an opposite end of thebody. The port is adapted to be pierced by a needle for withdrawal ofthe liquid sample. The open end is heat sealed after the liquid sampleha been introduced into the vessel. The auto-nucleating device isaffixed to the interior of the tubular body offset from the inlet sothat it cannot be contacted by a needle piercing the closed port.

In another embodiment, the cryopreservation device comprises a containerfor receiving and storing a liquid sample, the container having an inletfitting opening into the container and an adaptor mounted to thefitting. The adaptor has a first tubular branch and a second tubularbranch, with the second tubular branch terminating in a tube engagingfitting. A septum closes the first tubular branch, in which the septumis adapted to be pierced by a needle. The cryopreservation device isfurther provided with a tube engaged at one end to the tube engagingfitting on the second tubular branch and a closure at the opposite endof the tube.

The closure for the second branch is initially a septum that may bepierced by a needle for introduction of the sample liquid into thevessel. The container may be initially at below-atmospheric pressure toenhance transfer of the sample liquid from a syringe into the vessel.Once the sample liquid has been transferred, the tube on the secondbranch is heat sealed and severed just above the tube engaging fittingto form a final closure for the second branch. The closed device maythen be subject to a freezing and thawing protocol. After thawing asyringe may be used to withdraw the sample liquid through the septum inthe first branch of the adaptor.

It is contemplated that the present invention will provide a simple andreproducible system for induction of ice and reduction of supercoolingin many different cell freezing applications. The invention contemplatesmethods and devices for the controlled extracellular induction of icecrystals during cryopreservation of cells and tissues via theconstruction of solid-state matrix devices where ice nucleation willoccur spontaneously.

The present invention poses several advantages over prior systems andmethods. Currently, most methods of inducing controlled ice nucleationare cumbersome, difficult to reproduce, and are many times over-looked,despite the large body of literature pointing to the enhancedfreeze-thaw survival of many cells and tissues when the technique isemployed. To date, the most commonly used methods have ranged fromsimply touching the side of a vial or straw with a chilled (usually to−196° C.) metal object or cotton swab, to elaborate devices designed tospay liquid nitrogen on a small area of the sample. However, even whenperformed under optimal conditions, mechanically seeding ice crystals inthis manner can result in a failure to induce a large enough ice crystalto allow full propagation throughout the extracellular solution, or, inlocalized cell damage and loss due to the enormous cooling ratesobserved in the portion of the sample closest to where the metal objector liquid nitrogen spray is being directed on the container.

One object of the invention is to provide cell cryopreservation methodsand devices that significantly facilitate the freezing of a sampleliquid. One benefit of the invention is that it greatly reduces thequantity of cells that are damaged during cryopreservation. Anotherbenefit is that it permits cryopreservation of low-motility and/or lowcount sperm samples that could not be preserved using prior techniques.

Another benefit of the present invention is that it providescryopreservation vessels that are closed systems but that are readilyaccessible for multiple sample storage subjected to differentfreeze/thaw regimes. Other benefits and objects of the invention willbecome apparent upon consideration of the following written descriptionand accompanying figures.

DESCRIPTION OF THE FIGURES

FIG. 1 is a view of an auto-nucleating device according to oneembodiment of the present invention.

FIG. 2 is a view of known cryopreservation vessels incorporating theauto-nucleating device shown in FIG. 1.

FIG. 3 a is a view of a flexible closed system vial for cryopreservationof liquid samples according to a further embodiment of the invention,with the vial shown in an initial condition for delivery of a sample.

FIG. 3 b is a view of the vial shown in FIG. 3 a, shown with the vialend sealed.

FIG. 4 is a perspective view of a cell cryopreservation device accordingto another embodiment of the invention.

FIG. 5 is a perspective view of an adaptor used in the device shown inFIG. 4.

FIG. 6 is an exploded view of the device shown in FIG. 4.

FIG. 7 is a perspective view of a cell cryopreservation device accordingto a further embodiment.

FIG. 8 is a side cross-sectional view of a top portion of the deviceshown in FIG. 7.

FIG. 9 is a perspective cross-sectional view of a bottom portion of thedevice shown in FIG. 7.

DESCRIPTION OF THE ILLUSTRATED EMBODIMENTS

For the purposes of promoting an understanding of the principles of theinvention, reference will now be made to the embodiments illustrated inthe drawings and described in the following written specification. It isunderstood that no limitation to the scope of the invention is therebyintended. It is further understood that the present invention includesany alterations and modifications to the illustrated embodiments andincludes further applications of the principles of the invention aswould normally occur to one skilled in the art to which this inventionpertains.

In one embodiment of the invention, an auto-nucleating device 10 isprovided, as shown in FIG. 1, which involves the use of compositionscapable of ice nucleation. In accordance with the present invention, anice nucleating composition 20 is bound to the inner surface 14 of ahollow open tube 12. In a preferred embodiment, the tube is formed ofplastic. A sufficient amount of the nucleating composition is introducedinto the tube to form a solid matrix within the tube while permittingliquid flow through the tube.

In a preferred embodiment, the nucleating composition is crystallinecholesterol. The use of sterol compositions, and especially cholesterol,is known in other fields, such as in chill water systems, as shown inU.S. Pat. No. 4,928,493. In these other uses, powdered compositions aredisposed within a container for exposure to water to assist in theformation of ice. As explained below, it was determined afterexperimentation that crystalline cholesterol was non-toxic to the samplecells and liquids being prepared for cryopreservation, such as blood,stem cell solutions and semen.

The ends 16 of the tube are sealed with a solution-permeable membrane18. In particular, the membrane is permeable to the cryopreservationliquid and impermeable to cells or tissue to be preserved. It isimportant to maintain separation and prevent direct contact between thecells/tissue and the ice nucleating composition. The membranes at eachend will also contain any cholesterol crystals that may dislodge fromthe tube and prevent the crystals from contaminating the surroundingliquid. It is also important that the membrane permit free flow of thecryopreservation liquid into the tube 12. The tube and the intersticesin the solid matrix nucleating composition may also be initially filledwith an isotonic buffer.

This auto-nucleating device 10 is sized to be placed into acryopreservation vessel such as a vial 30 or a blood bag 40, as shown inFIG. 2. The device may be free within the vessels or may be affixed toan interior surface. Since the device is intended as a nucleation sitefor ice formation, it does not need to be very large. In a specificembodiment, the tube 12 is 0.25 inches long and 0.0625 inches indiameter. The cryopreservation vessel may be filled with the particularspecimen or sample, and a cryopreservation solution, where appropriate,as is known in the art, while the device 10 remains within thecontainer. The container 30 or 40 is then subjected to acryopreservation protocol. Since the cryopreservation liquid is incontact with the ice nucleating composition 20 within the device, icewill spontaneously form inside the tube 12 of the device 10 with littleor no supercooling. Ice then continues to build off the tube into thesurrounding solution, resulting in freezing of the cell suspension withlittle or no supercooling and minimal intracellular ice formation.

In one specific embodiment, a first experiment was designed to determinethat cholesterol physically bound to the inside of cryo-storage vesselswill induce ice nucleation. In this embodiment, the working sterolsolution was prepared by adding 0.025 g of dry cholesterol to 3 ml ofmethanol. The resulting suspension was then placed into a 70° C. drybath and agitated intermittently until all solid sterol had dissolved.Commercially available vials were coated with 100 μl of the sterolsolution and placed in the dry bath at 75° C. to allow the methanol toevaporate, and to achieve cholesterol recrystallization and adhesion.Vials were then rinsed with 1 ml of PBS, 2-3 times, to remove any loosecrystals.

Next, solutions of 6% glycerol (to replicate a typical sperm bankcryopreservation media) and 10% DMSO (to replicate a generalizedcell-line cryopreservation system) were prepared in PBS and wereevaluated by cooling at 5° C./minute in a sterol coated vial and in anon-coated (control) vial. To achieve statistical power, 20 vialscontaining DMSO and 12 vials containing glycerol were evaluated. Thetemperature inside each vial was monitored using a thermocouple at onesecond intervals to allow resolution of the solution freezing point andrelease of the latent heat of fusion.

The results of this experiment indicated that in both DMSO and glycerolthe freezing point was higher and the temperature change during heat offusion (ΔT) was reduced for vials coated with the sterol. These resultsare summarized in the following table:

FREEZING POINT ΔT Sterol Coated; DMSO −4.56 ± 1.72° C. 0.37 ± 1.29° C.Non-Coated; DMSO −10.61 ± 3.52° C.  5.63 ± 4.36° C. Sterol Coated;Glycerol −2.97 ± 1.14° C. 1.54 ± 1.47° C. Non-Coated; Glycerol −9.33 ±4.01° C. 7.24 ± 3.61° C.

In this experiment, some sloughing or chipping of the crystals (and somedegree of dissolution in the DMSO samples) was also observed, resultingin solution contamination (possibly due in part to unavoidable physicalmanipulation of the containers and the solutions). In order to addressthis problem, one embodiment of an auto-nucleating device 10 wasprovided in which a 0.25 inch hollow tube was coated on the interiorwith 100 μl of sterol solution and allowed to dry for 48 hours. One endof the tube was sealed with a permeable cotton plug, while the other endof the tube was attached to the inside of a vial lid using an epoxyresin and allowed to dry for 14-24 hours. The stent was designed to keepthe bound cholesterol in a sequestered environment while still allowingsolution (but not cells) in to make contact.

In a second experiment, human semen was cryopreserved using thisauto-nucleating device 10 and was specifically analyzed to determinewhether the samples cryopreserved in accordance with the presentinvention had a higher post-thaw viability than semen frozen usingstandard configuration vials. In this experiment, discarded human semensamples (20 samples from 4 donors) were obtained and were placed into ahumidified 37° C. incubator (5% CO₂, 95% air) for 30-60 minutes untilliquefied. Once liquefied, the samples were adjusted to 5 m1 usingisotonic PBS (equilibrated to 37° C.) and evaluated using a computerassisted semen analysis device to measure and record overall initialcount and motility. The samples were then equilibrated to 6% glycerol ina TEST egg yolk buffer through a step-wise addition procedure. Followingequilibration, each sample was divided into three 1.5 ml aliquots anddeposited into (1) a vial containing the device 10; (2) a standard vialto be manually seeded (positive control); and (3) a standard vial whichwas to receive no seeding (negative control).

All samples were placed into a controlled-rate freezer and cooled from22° C. to −8° C. at −5° C./min. After 3 minutes at −8° C., a cotton swabthat had been soaked in liquid nitrogen was used to initiate seeding inthe manually seeded vial. After an additional 7 minutes at −8° C.,specimens were cooled again at −10° C./minute down to −40° C. At −40° C.the rate was increased to −20° C./minute, and at −80° C. samples wereplunged into liquid nitrogen (LN₂).

Following freezing, the samples were thawed by placing on the bench top(corresponding to ˜300° C./min thawing rate). Once the last of the icehad melted, the glycerol was then diluted drop-wise over a 10-minuteperiod by the addition of PBS; samples were then washed and re-suspendedin glycerol-free PBS. Finally, samples were incubated (37° C.,humidified atmosphere, 5% CO₂, 95% air) for at least one hour prior toevaluation of post thaw count and motility.

The results of this second experiment indicated that samples frozenusing the auto-nucleating device of the present invention retainedsignificantly (p<0.05) higher motility (66.1±4.7% mean±SEM) than thosefrozen using manual seeding (56.0±3.8%). Both seeding approaches weresignificantly higher (p<0.05) than the unseeded, negative controlsamples (43.4±3.7%) as determined using analysis of variance techniques.

In a third experiment it was determined that bound cholesterol wouldproduce no cytotoxic effects on semen cultured over an extended periodof time. In this experiment, liquefied semen samples were exposed toculture plates that had been coated with 100μ1 of the sterol solution.Motility evaluations performed at 1, 2, 4 and 8 hours of incubationshowed no significant cytotoxic effect of direct contact with boundcholesterol on human spermatozoa over 8 hours of culture.

Thus, the auto-nucleation device 10 of the present invention isdemonstrated to yield better post-thaw motility than using either manualseeding or no seeding in sperm cryopreservation procedures. Theseexperiments demonstrated that sterol-induced ice nucleation is aconsistent, reliable method which can reduce supercooling and thereforereduce the associated rapid increase in temperature following the“flash” of ice crystal formation typical of the supercooled solutionfreezing event with better outcomes. The device and method of thepresent invention allows the samples to remain in the cooling chamberundisturbed throughout the entire duration of freezing because there isno need for the manual seeding techniques of the prior art.

It is believed that the device and methods of the present invention areparticularly suited for standard, commercial sperm banking methods. In astandard commercial sperm bank setting many samples are processed andtime/staff constraints do not always allow for controlled rate coolingor for the careful handling that can be achieved in the laboratory. Itis believed that the present invention permits repeatablecryopreservation of samples with outcomes that exceed currenttechniques. In addition, the present invention can enable successfulfreezing and recovery of samples with low motility that would normallybe excluded from donor pools.

Similarly, the device 10 and methods of the present invention may havesignificant impact on the ability to store cryopreserved hematopoieticstem and/or progenitor cells (PCB HPCs) in a manner that allows forbanking and sufficient time for adequate infectious disease screening aswell as HLA typing to be performed. Cryopreservation offers theopportunity for preserving PCB derived HPCs from neonatal patients whomay benefit from gene therapy, or who are at risk of loosing normalhematopoietic function through disease or iatrogenically via radio-and/or chemotherapy. Recently, increasing efforts have been directedtoward refining progenitor cell selection methods. The ability topreserve these relatively “pure” progenitor cell populations (e.g. cellsexpressing the CD34 surface glycoprotein) potentially minimizes thetotal volume of the transplanted cell suspension. However, because thevolume of PCB typically acquired is much smaller than bone marrowsamples, limited numbers of HPCs per kilogram recipient weight can beobtained. This makes efficient and optimal cryopreservation methods forPCB derived HPCs much more critical than in the case of other sources ofHPCs (e.g. bone marrow, peripheral blood). The auto-nucleation device ofthe present invention produces more efficient and optimal means forcryopreservation and recovery of such delicate samples than hasheretofore been available. It is believed that integration of the device10 into ongoing research and development of improved cord blood stemcell cryopreservation methods will result in a unique approach topreserving this cell type with higher recovery with less labor.Experimental protocols have been developed to verify the viability ofthe device and methods of the present invention in the cryopreservationof PCB and cord blood, as well as bull semen used in commercialartificial insemination facilities. These protocols are described in theabove-referenced provisional application No. 60/814,982, whichdescription is incorporated herein by reference.

A further aspect of the present invention recognizes thatcryopreservation of various cord blood derived stem/progenitor cells mayrequire completely different procedures and therefore different storagecontainers than exist under currently known procedures. For banking andstorage of multiple cell types derived from umbilical cord blood, it maybe optimum to use very different freezing protocols including differentcooling/warming rates. Current technology relies either on cryogenicbags, some with multiple chambers, or vials. However both of thesesystems have substantial drawbacks. The multiple chamber bags do notallow for different cooling rates or CPAs to be used in the differentchambers. Vials by themselves cannot be considered “closed” systems atcryogenic temperatures unless a heat sealed over wrap is used, theapplication of which can compromise sensitive samples.

To overcome this limitation, a further embodiment of the inventionresides in a cryopreservation vessel in the form of a flexible closedsystem vial 50, illustrated in FIGS. 3 a, b, which allows the sample tobe split between separate units and frozen using different protocols ina closed system. The vial 50 includes a flexible tubular body 52 havinga port 54 at one end. The port is sealed, preferably by the samematerial as the flexible body, but is adapted to be punctured by aneedle for aseptically withdrawing the sample after thawing.

As shown in FIG. 3 a, the opposite end 56 of the vial is initially opento permit introduction of a liquid sample. Once the vial 50 has beenfilled, the end 56 is closed, such as by a heat seal strip 58, as shownin FIG. 3 b. The closed system vial 50 is then available for freezingand storage of a single unit. Optionally, but preferably, each vialincludes the auto-nucleation device 10 described above. As shown in FIG.3 a, the device 10 is preferably adhered to the inner wall of the body52 so that it is not accessible by a needle passing through the port 52.

It is contemplated that the vial 50 of the present embodiment may beused for multiple freeze/thaw protocols in discrete cryo-containers.Thus, an array of vials 50 may be supported in a fixture with the openend 56 available for introduction of multiple aliquots of the liquidsample. When each vial is filled, the corresponding end is sealed toprovide a closed system vial for cryopreservation.

In a further embodiment of the invention, a cryopreservation device 60is provided, as shown in FIGS. 4-6, that further simplifies the processof obtaining a sample and preparing it for freezing. The device includesa container 62 sized to receive the liquid sample. The container 62includes an inlet fitting 64 at one end. As shown in FIG. 6, anauto-nucleation device 10 may be introduced into the container throughthe inlet fitting 64.

The inlet fitting receives an adaptor 65, shown in detail in FIG. 5. Theadaptor includes a lower tubular portion 66 that is sized to fit snuglywithin the inlet fitting 64. The lower portion 66 may be sealed to theinlet fitting using an epoxy or heat sealing, or other suitable meansfor providing an air and liquid-tight seal between the container 62 andthe adaptor 65.

The adaptor includes two tubular branches 67 and 69. The branch 67terminates in an end portion 68 that is configured to engage a needleseptum 72 (FIG. 6). The second branch 69 terminates in a barbed fitting70. This barbed fitting 70 is in sealed engagement with the end 74 a oftubing 74. The free end 74 b of the tubing 74 receives its own needleseptum 75. Both needle septums 72 and 75 are configured to provide anair and liquid-tight seal at the end of the two branches 67, 69.Moreover, the septums 72, 75 are configured to be pierced by a needle ina known manner and are self-sealing once the needle is removed.

In one specific embodiment, a tubing clip 80 is provided to stabilizethe tubing 74 when it is engaged to the adaptor 65. The clip 80 includesa portion 80 configured to slide over the branch 67 of the adaptor andan attached portion 84 that is configured to slide over the tubing 74,as shown in FIG. 4.

The container 62 of the cryopreservation device 60 is sized to bereceived in the standard “egg carton” separator used to transfer andstore cell samples for freezing and eventual thawing. It is contemplatedthat several such cryopreservation devices 60 carrying cell samples froma common source may be housed in a common egg carton separator. In use,the device 60 is initially stored in the configuration shown in FIG.4—i.e., with the tubing 74 projecting upward from the cell containeritself. The adaptor 65 is sized so that it does not extend beyond thevertical envelope of the container and therefore will not interfere withthe storage of other like devices 60. The tubing 74 is shown with a bendthat extends outside the vertical envelope. If the devices in the eggcarton container are properly aligned, the tubing 74 will not interferewith other cell containers. However, in accordance with the preferredembodiment, it is contemplated that the tubing 74 will be flexible sothat it can be arranged as necessary to avoid interfering with othercontainers 62 in the same egg carton separator.

The tubing 74 is preferably flexible for an additional reason. Inparticular, the branch 69 and the attached tubing 74 is used for fillingthe container 62 of the device 60. Thus, in accordance with the presentinvention, the flexible tubing 74 may be manipulated to permitintroduction of a newly extracted cell sample into the container. Thisintroduction occurs in one aspect by piercing the septum 75 with aneedle of a syringe containing the extracted liquid sample.Alternatively, the septum 75 may be removable from the end 74 b of theflexible tubing so that the sample may be injected directly into thetubing without having to pierce a membrane. In either case, the flexibletubing 74 facilitates this step of filling the container 62 since thetubing can be manipulated as necessary while the container remains inthe egg carton container.

Once the sample has been introduced into the container 62 it iscontemplated that the branch 69 of the adaptor is permanently sealed. Inthe preferred embodiment, this sealing occurs by sealing the flexibletubing just above the barbed fitting 70. Once sealed, the remainder ofthe tubing can be removed since it is no longer needed. In one specificembodiment, a known pinch sealing bar may be used to simultaneouslyflatten the tubing, heat seal the flattened portion and sever the excessportion. This sealing and cutting preferably occurs as close to thebarbed fitting 70 as possible so that no remainder of the flexibletubing 74 will fall outside the vertical envelope of the container 62.

It is desirable that the sealing and cutting steps not compromise thesterile integrity or closed, sealed aspect of the cryopreservationdevice 60. When the sample is injected through the septum 75 the branch69 remains sealed throughout the process, even after the needle isremoved. Once the branch 69 is sealed the device 60 containing theliquid sample is ready for freezing and storage in the same egg cartonthat housed the device during the filling step. When it is desired toretrieve the sample, the device 60 may be removed from the egg cartonfor individual thawing apart from the other devices held in the carton.The needle septum 72 of branch 67 provides the avenue for sterilewithdrawal of the sample. Thus, a needle and syringe may be used topierce the septum and withdraw the liquid sample into the syringe. Theempty device 60 may then be discarded.

In an alternative embodiment, a cryopreservation device 100 may beprovided as illustrated in FIGS. 7-9. The device includes a container102 that is similar to the container 62 described above in itscapability for cryostorage of a cell suspension, with or without anauto-nucleating device 10 disposed therein. In certain embodiments, thecontainer 100 has a storage volume of 2-5 mL. The container 102 has anopen top portion 103 and a bottom portion 104 defining an opening 105.The top portion 103 is hermetically sealed by a cap 110, as shown in thecross-sectional view of FIG. 8. The top portion defines an annularflange 107 over which is mounted a lower skirt 116 of the cap 110. Thislower skirt is preferably heat sealed to the annular flange inconventional manner to form a fluid-tight joint capable of withstandingcryogenic temperatures.

The cap 110 supports an inlet branch 112 and a vent branch 114, each ofwhich may be in the form of sterile tubing. The inlet branch 112 may beprovided with a standard needle septum, such as the septum 75 describedabove. The two branches 112, 114 are mounted over corresponding fittings118, 120, with each fitting defining a passageway 119, 121 incommunication with the interior of the container 112. Each branch ishermetically sealed over the corresponding fitting, again to form afluid-tight cryogenic-capable joint.

In the embodiment of FIGS. 7-9, the branch 114 serves as a vent branch,rather than as an outlet branch as in the previous device 60. A filterelement 125 may be disposed within the passageway 121 of the fitting120. In a preferred embodiment, this filter element is a 3 μm sterilemicro-filter. The micro-filter 125 is gas permeable but generallyimpermeable to the liquid sample or cell suspension being stored withinthe container.

In the present embodiment, the device 100 provides for removal of thestored suspension through the bottom portion 104 of the container 102,rather than through the cap as in the prior embodiment. Thus, as shownin the cross-sectional view of FIG. 9, the opening 105 in the containeris sealed by a needle septum 130. The outlet septum 130 is suitablyhermetically sealed to the container 102, such as by heat sealing. Toprotect the septum, a removable cover 132 may be provided that isaffixed to the container around the septum. The cover 132 may define atear portion 133 in the form of a thinner or weakened region that allowsthe central portion 135 of the cover to be removed. A tab 135 may beprovided, as shown in FIG. 7, to facilitate removal of the centralportion 135. The cover may be a foil film that is adhered around itsperimeter to the bottom portion 104 of the container 102.

The manner of use of the cryopreservation device 100 is similar to thedevice 60 described above. In particular, a cell suspension isintroduced into the container 102 through the inlet branch 112, andparticularly through the needle septum described above. Alternatively,the inlet branch may be provided with a standard needle or needle-lessport. As the cell suspension is injected into the container, the airwithin is vented through the filter 125 in the vent branch 114. Incertain embodiments, the micro-filter 125 is configured to be generallyimpermeable to the cell suspension. Thus, once the level of thesuspension within the container reaches the filter 125 the suspensionwill not pass through the filter and the increase in pressure willterminate the flow of liquid into the container.

Once the container 102 is filled the two branches may be heat sealed,using for instance the heated pinch sealer described above, so that thecontainer is completely and hermetically sealed. Of course it isunderstood that the needle septum 130 and cover 132 remain intact as thecontainer 102 is filled. The filled and sealed device 100 can then becryogenically stored.

When it is desired to withdraw the cell suspension, the device andcontents are first thawed in a known manner. Once thawed, the cover 132is removed to expose the septum 130. The vent branch 113 is severed toopen the vent passageway 121. The cell suspension may then be withdrawnby a syringe or similar device puncturing the septum 130. The bottomportion 104 of the container may define a series of prongs 106 that areadapted to engage the withdrawal device or syringe. In some embodimentsthe prongs 106 are configured for docking the withdrawal device, whilein other embodiments the prongs may be provided with LUER threads forengaging the LUER fitting on the withdrawal syringe.

It is contemplated in some embodiments that the containers 62 and 102 ofthe cryopreservation devices 60 and 100 may be provided with an initialvacuum. This vacuum assists withdrawal of the liquid sample during thestep of filling the device containers 62, 102. Since the openings toeach branch 67, 69 and 112, 114 are sealed (either by the correspondingseptums 72, 75 or by heat-sealing), the vacuum may be maintained over along period of time. A cap may be provided over each branch to ensure anair-tight seal. In a specific embodiment, the initial vacuum in thecontainers 62, 100 may be at a sub-atmospheric pressure of between 100mmHg (absolute) and about 160 mmHg (absolute).

The cryopreservation devices 60 and 100 may be formed of standardmaterials used in the field of blood banking and long-term storage instandard cryogenic conditions (i.e., temperatures as low as −196° C.).In order to fit in standard egg carton containers, the device 60 (aftersealing of the flexible tubing) should fit within a 10 mm diameter and a90 mm height. The flexible tubing 74 must also be capable ofwithstanding cryogenic temperatures without compromising the ability toheat seal and sever the tubing when sealing he branch 69 of the adaptor65. In one specific embodiment, the flexible tube is formed of TYGON® ora similar material.

While the invention has been illustrated and described in detail in thedrawings and foregoing description, the same should be considered asillustrative and not restrictive in character. It is understood thatonly the preferred embodiments have been presented and that all changes,modifications and further applications that come within the spirit ofthe invention are desired to be protected.

What is claimed is:
 1. A method, comprising: cryogenically storing acryopreservation container having a chamber defined between a firstopening and a second opening and a cellular composition received in thechamber, the cryopreservation container further including a first sealedtubular branch communicating with said first opening, a second sealedtubular branch communicating with said first opening, and a septumclosing said second opening of said container, said septum adapted to bepierced by a needle, a removable cover operable to protect said septumwhen subjected to cryopreservation conditions, and a support cap havinga lower portion hermetically sealed to said container, said support capalso comprising an upper portion configured to support said first sealedtubular branch and said second sealed tubular branch.
 2. The method ofclaim 1, also comprising: after said cryogenically storing, subjectingthe cryopreservation container to conditions sufficient to thaw thecellular composition.
 3. The method of claim 2, also comprising:piercing the septum with a needle; and withdrawing an amount of thecellular composition through the needle.
 4. The method of claim 1,wherein the first and second sealed tubular branches each include aheat-sealable tube material having a heat-sealed region of the tubematerial.
 5. The method of claim 1, wherein said cryopreservationcontainer further comprises a gas-permeable sterile micro-filter withinsaid second sealed tubular branch.
 6. The method of claim 5, whereinsaid gas-permeable sterile micro-filter is configured to be generallyliquid impermeable.
 7. The method of claim 1, wherein saidcryopreservation conditions include temperatures as low as −196° C. 8.The method of claim 1, wherein said cover comprises a foil film.
 9. Themethod of claim 1, further comprising: removing said cover prior towithdrawing an amount of the cellular composition through said septum.10. The method of claim 1, wherein said cryopreservation container isformed of a plastic suitable for storing a blood sample.
 11. The methodof claim 1, wherein said cryopreservation container is formed of aplastic suitable for storing a liquid sample at cryogenic temperaturesas low as −196° C.
 12. The method of claim 1, further comprising:severing said second sealed tubular branch prior to withdrawing anamount of the cellular composition.
 13. A method, comprising:cryogenically storing a cryopreservation container having a chamberdefined between a first opening and a second opening and a cellularcomposition received in the chamber, the cryopreservation containerfurther including a first sealed tubular branch communicating with saidfirst opening, a second sealed tubular branch communicating with saidfirst opening, and a septum closing said second opening of saidcontainer, said septum adapted to be pierced by a needle, a removablecover operable to protect said septum when subjected to cryopreservationconditions, and an adaptor body mounted to said first tubular branch andsaid second tubular branch, said adaptor body having an inlet tubularbranch and a vent tubular branch.
 14. The method of claim 13, alsocomprising: after said cryogenically storing, subjecting thecryopreservation container to conditions sufficient to thaw the cellularcomposition.
 15. The method of claim 14, also comprising: piercing theseptum with a needle; and withdrawing an amount of the cellularcomposition through the needle.
 16. The method of claim 13, wherein thefirst and second sealed tubular branches each include a heat-sealabletube material having a heat-sealed region of the tube material.
 17. Themethod of claim 13, wherein said cryopreservation container furthercomprises a gas-permeable sterile micro-filter within said second sealedtubular branch, and wherein said gas-permeable sterile micro-filter isconfigured to be generally liquid impermeable.
 18. The method of claim13, wherein said cryopreservation conditions include temperatures as lowas −196° C.
 19. The method of claim 13, further comprising: removingsaid cover prior to withdrawing an amount of the cellular compositionthrough said septum.
 20. The method of claim 13, further comprising:severing said second sealed tubular branch prior to withdrawing anamount of the cellular composition.